The present invention relates to certain derivatives of 4-demethylpenclomedine (also referred to herein as DM-PEN) and especially to acyl derivatives of DM-PEN. The present invention also relates to pharmaceutical compositions comprising the acyl derivatives of the present invention, as well as a method of using the compounds in treating cancer in a mammal. The present invention also relates to a method for producing the compounds of the present invention.
Even though significant advances have occurred in treatment of cancer, it still remains a major health concern. It has been reported that cancer is the cause of death of up to one of every four Americans.
Included among the known chemotherapeutic drugs are carmustine, doxorubicin, methotrexate, TAXOL(copyright), nitrogen mustard, procarbazine, and vinblastine, to name only a few. However, many chemotherapeutic drugs also produce undesirable side effects in the patient. For example, U.S. Pat. No. 4,717,726 reportedly discloses a compound suitable for inhibiting the growth of certain types of malignant neoplasms in mammals. See also Plowman et al., Cancer Res., 49 (1989), 1909-1915. The disclosed compound, 3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine, also known as penclomedine, is not satisfactory as a chemotherapeutic, however, because it is known to produce certain undesirable side effects especially in the central nervous system.
For example, neurological and hematological toxicities of penclomedine have been reported in preclinical and early clinical studies. Dose related neurotoxicity, consisting of muscle tremors, incoordination, convulsions and reduced activity, has been observed in rats. Neurotoxicity appears to be related to peak plasma drug concentrations, as it developed during or immediately after infusion and could be ameliorated by decreasing the rate of drug administration. In dogs, severe emesis and seizures have been associated with plasma penclomedine levels above 30 xcexcM. Neurotoxicity, consisting of dysmetria, ataxia, and vertigo, was also the principal dose limiting toxicity of penclomedine administered as a one hour infusion for 5 consecutive days in patients with advanced solid tumors. The presence of these toxicities, at much lower peak plasma concentrations compared to those reported in preclinical studies, may preclude the administration of higher doses of penclomedine and the achievement of concentrations associated with optimal antitumor activity. Berlin et al., Proc. Amer. Assoc. Cancer Res., 36, 238 (1005); O""Reilly et al., Proc. Amer. Soc. Clin. Oncol., 14, 471 (1995).
Some relevant background art can be found in O""Reilley et al., Clinical Cancer Research, 2 (March 1996), 541-548. This reference describes a study to assess the distribution of 14C-penclomedine in the tissues and tumors of tumor-bearing rats. The study found that the predominant radioactive species in the brain was penclomedine, which may explain the observed neurotoxicity of the drug.
More recently, 3,5-dichloro-2-methoxy-4-hydroxy-6-(trichloromethyl)pyridine or 4-demethylpenclomedine has been suggested as a compound for treating cancer. See WO 97/46531 to Hartman et al.
Notwithstanding the advances in cancer treatment that have been made, there still remains room for improved drugs that are effective in treating cancer, while at the same time exhibit reduced adverse side effects.
The present invention relates to novel acyl derivatives of 4-demethylpenclomedine represented by the following formula I: 
and pharmaceutically acceptable salts thereof.
Another aspect of the present invention relates to pharmaceutical compositions containing the above-disclosed compounds.
The present invention is also concerned with methods of using the compounds of the present invention in treating cancer in a mammal.
A still further aspect of the present invention is concerned with a method for preparing the above-disclosed compounds of the present invention.
In particular, acyl compounds of the present invention can be produced by reacting 4-demethylpenclomedine with an acylating agent such as an acyl chloride or acyl anhydride.
If desired, such reaction can be carried out in the presence of a base.
Still other objects and advantages of the present invention will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described only the preferred embodiments of the invention, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.
The present invention is concerned with novel acyl derivatives of 4-demethylpenclomedine compounds represented by the formula: 
and pharmaceutically acceptable salts thereof.
The acyl groups can be straight or branched chained, can be unsubstituted or substituted such as with halogen such as Cl, Br and I, and/or include 5 and 6 membered rings. The ring moiety can be a carbocycle or a heterocycle including a hetero atom such as O, S or N. Typically, the acyl group contains 1-12 carbon atoms.
Examples of such suitable acyl groups are the following, each of which has been evaluated for activity according to the present invention, and each has been characterized by mass spectroscopy: 
Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulfonic, for example p-toluenesulfonic acid.
It has been found according to the present invention that the acyl compounds of the present invention are surprisingly and advantageously useful in treating mammalian cancer, especially human cancer. The compounds of the present invention have been shown to exhibit generally superior activity in comparison to 4-demethylpenclomedine and penclomedine. Moreover, the compounds of the present invention are believed to possess reduced toxicity in comparison to both demethylpenclomedine and penclomedine (PEN).
It is further noted, as will be discussed below, that the acyl compounds of the present invention are not considered to be prodrug forms of DM-PEN. Both penclomedine (PEN) and DM-PEN are inactive as cytotoxic agents in vitro but must be metabolized to produce cytotoxicity, as demonstrated by their anticancer activity in vivo, which indicates that DM-PEN, as well as PEN, is a prodrug of the ultimate activated metabolite. The proposed mechanism by which PEN and DM-PEN exhibit cytotoxicity in vivo is shown in Scheme 1 and indicates that PEN and DM-PEN are on the same metabolic activation pathway, which includes a non-acylated, free radical activated for DNA crosslinking. In contrast, it is believed (Scheme 2) that the acyl derivatives (DM-ACYL-PEN) are converted via the liver to an acylated free radical (rather than a non-acylated free radical) and, as such, are not fully activated for DNA crosslinking but remain in a prodrug form as they exit the liver and enter the circulation. The partially activated acylated free radical is transported to cells via circulation and forms an adduct with nuclear DNA. Subsequently, the adduct is deacylated by general cellular esterases particularly in the tumor cell, which allows for in situ, full activation and possibly reduced generalized host toxicity in comparison to PEN and DM-PEN, both of which are fully activated in the liver for DNA adduction and crosslinking before being transported via circulation to tumor and non-tumor cells. Deacylation generates a 4-hydroxy moiety on the pyridine nucleus, which then tautomerizes via the enol-keto mechanism to produce an alpha-haloketo moiety at the 4,5-positions in which the 5-chloro function is activated for displacement by a nucleophilic moiety on the DNA for completion of a DNA crosslink, as shown in Scheme 2. This mechanism is further supported by the generally superior activity of the acyl derivatives in comparison to DM-PEN vs. both subcutaneously (s.c.)- and intracerebrally (i.c.)-implanted human MX-1 mammary tumor xenograft, s.c.-implanted human U251 CNS tumor xenograft, and both parent, and particularly, cyclophosphamide (CPA)-resistant lines of P388 leukemia, as shown in Tables 1-4 below. 
A general experimental procedure for preparation of acyl derivatives of DM-PEN is as follows: DM-PEN (1 g) in 15 ml dry dichloromethane is treated with 0.5 ml triethylamine followed by one equivalent of an acyl chloride added dropwise at room temperature in 5 ml dry dichloromethane. The solution is stirred 30 min at room temperature and evaporated to dryness via a water aspirator. The residue is triturated with 5 ml acetone and filtered to remove triethylamine hydrochloride. The acetone filtrate is concentrated to 1 ml and separated on an 8 inch, 2 mm silica gel plate containing a fluorescent indicator. The major UV-visible band is eluted with acetone and the solvent evaporated, giving the DM-ACYL-PEN product in high yield. Characterization is provided by fast-atom-bombardment mass spectrometry (FABMS), which reveals the appropriate mass number +1 corresponding to the expected structure, and thin-layer chromatography, which yields a single UV-visible component.
Although DM-PEN can be used as the starting material for the 4-acyl derivatives, synthesis of DM-A-PEN is also accomplished by the following route that does not include DM-PEN as an intermediate: and methods disclosed in U.S. patent application Ser. No. 60/116,676 entitled xe2x80x9cPyridine Compounds, Use and Preparation Thereofxe2x80x9d, filed concurrently herewith, the entire disclosure of which being incorporated herein by reference. 
As illustrated above, the acyl compounds of the present invention can be produced by reacting 3,4,5,6-tetrachloro-2-(trichloromethyl)pyridine with an alcohol in the presence of a base to produce the corresponding 3,5-dichloro-4,6-dialkoxy-2-(trichloromethyl) pyridine, which in turn is reacted with a dealkylating agent such as anhydrous aluminum chloride to produce didemethylpenclomedine.
Of course, other dealkylating agents can be used if desired. The didemethylpendomedine is then reacted with an acylating agent to form the corresponding 4-acyl-6-demethylpenclomedine, which is then reacted with an alkylating agent such as (trimethylsilyl)diazomethane to produce the desired acyl derivative.
It is understood that other acylating agents and alkylating agents can be used. Moreover, various reaction parameters such as temperature, relative amounts, and pressure can be selected by those skilled in the art once aware of the present disclosure.
Novel precursors for producing the acyl derivatives of the present invention are represented by the following formulae: 
wherein
each R1 is independently an alkyl group provided that at least one R1 is an alkyl group containing at least two carbon atoms; and
each R2 is independently H or acyl.
The alkyl group typically contains 1 to 22 carbon atoms and preferably 2 to 22 carbon atoms.
The R2 acyl groups can be straight or branched chained, can be unsubstituted or substituted such as with halogen such as Cl, Br and I, and/or include 5 and 6 membered rings. The ring moiety can be a carbocycle or a heterocycle including a hetero atom such as O, S or N. Typically, the acyl group contains 1-12 carbon atoms.
Although many of the above precursor compounds do not exhibit any appreciable anti-cancer activity, it has been observed that at least a few of the precursor compounds show active anti-cancer activity. These compounds have the structure wherein at least the R1 group located at position 4 is ethyl and include 3,5-dichloro-4,6-diethoxy-2-(trichloromethyl)pyridine and 3,5-dichloro-4-ethoxy-6 methoxy-2-(trichloromethyl) pyridine. Therefore, those compounds wherein at least the R1 group at position 4 is ethyl or pharmaceutically acceptable salts thereof can be used in treating cancer in a mammal. However, these precursor compounds are not as active as the acyl compounds of the present invention and also exhibit higher toxicity as compared to acyl compounds of the present invention.